title: Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae.
abstract: Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.
authors.affiliation: Universität Regensburg, Biochemie-Zentrum Regensburg (BZR), Lehrstuhl für Biochemie III, 93053 Regensburg, Germany and Department of Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hamperl
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Brown
authors.forename: Ana Villar
authors.initials: AV
authors.lastname: Garea
authors.forename: Jorge
authors.initials: J
authors.lastname: Perez-Fernandez
authors.forename: Astrid
authors.initials: A
authors.lastname: Bruckmann
authors.forename: Katharina
authors.initials: K
authors.lastname: Huber
authors.forename: Manuel
authors.initials: M
authors.lastname: Wittner
authors.forename: Virginia
authors.initials: V
authors.lastname: Babl
authors.forename: Ulrike
authors.initials: U
authors.lastname: Stoeckl
authors.forename: Rainer
authors.initials: R
authors.lastname: Deutzmann
authors.forename: Hinrich
authors.initials: H
authors.lastname: Boeger
authors.forename: Herbert
authors.initials: H
authors.lastname: Tschochner
authors.forename: Philipp
authors.initials: P
authors.lastname: Milkereit
authors.forename: Joachim
authors.initials: J
authors.lastname: Griesenbeck
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics; D015825:Chromosomes, Fungal / Q000737:chemistry*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D023281:Genomics / Q000379:methods; D006657:Histones / Q000378:metabolism; D013058:Mass Spectrometry; D009707:Nucleosomes / Q000737:chemistry; D011401:Promoter Regions, Genetic; D020543:Proteome / Q000302:isolation & purification; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004275:DNA, Ribosomal; D006657:Histones; D009707:Nucleosomes; D020543:Proteome; D012341:RNA, Ribosomal, 5S; D029701:Saccharomyces cerevisiae Proteins; D000135:Acid Phosphatase; C488916:PHO5 protein, S cerevisiae